Novel chromophores/fluorophores and methods for using the same

ABSTRACT

Nucleic acid compositions encoding novel chromo/fluoroproteins and mutants thereof, as well as the proteins encoded the same, are provided. The proteins of interest are proteins that are colored and/or fluorescent, where this feature arises from the interaction of two or more residues of the protein. The subject proteins are further characterized in that they are either obtained from non-bioluminescent  Cnidarian , e.g.,  Anthozoan , species or are obtained from  Anthozoan  non- Pennatulacean  (sea pen) species. Specific proteins of interest include the following specific proteins: hcriGFP; dendGFP; zoanRFP; scubGFP1; scubGFP2; rfloRFP; rfloGFP; mcavRFP; mcavGFP; cgigGFP; afraGFP; rfloGFP2; mcavGFP2; and mannFP. Also of interest are proteins that are substantially similar to, or mutants of, the above specific proteins. Also provided are fragments of the nucleic acids and the peptides encoded thereby, as well as antibodies to the subject proteins and transgenic cells and organisms. The subject protein and nucleic acid compositions find use in a variety of different applications. Finally, kits for use in such applications, e.g., that include the subject nucleic acid compositions, are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of International ApplicationSerial No. PCT/US02/36499 filed on Nov. 12, 2002 and designating theUnited States; which application (pursuant to 35 U.S.C. § 119 (e))claims priority to the filing date of U.S. Provisional PatentApplication Ser. No. 60/332,980 filed Nov. 13, 2001; the disclosure ofwhich is herein incorporated by reference.

INTRODUCTION

1. Field of the Invention

The field of this invention is chromoproteins and fluorescent proteins.

2. Background of the Invention

Labeling is a tool for marking a protein, cell, or organism of interestand plays a prominent role in many biochemistry, molecular biology andmedical diagnostic applications. A variety of different labels have beendeveloped, including radiolabels, chromolabels, fluorescent labels,chemiluminescent labels, etc. However, there is continued interest inthe development of new labels. Of particular interest is the developmentof new protein labels, including chromo- and/or fluorescent proteinlabels.

Relevant Literature

U.S. Patents of interest include: 6,066,476; 6,020,192; 5,985,577;5,976,796; 5,968,750; 5,968,738; 5,958,713; 5,919,445; 5,874,304; and5,491,084. International Patent Publications of interest include: WO00/46233; WO 99/49019; and DE 197 18 640 A. Also of interest are:Anderluh et al., Biochemical and Biophysical Research Communications(1996) 220:437-442; Dove et al., Biological Bulletin (1995) 189:288-297;Fradkov et al., FEBS Lett. (2000) 479(3):127-30; Gurskaya et al., FEBSLett., (2001) 507(1):16-20; Gurskaya et al., BMC Biochem. (2001) 2:6;Lukyanov, K., et al (2000) J Biol Chemistry 275(34):25879-25882; Maceket al., Eur. J. Biochem. (1995) 234:329-335; Martynov et al., J. Biol.Chem. (2001) 276:21012-6; Matz, M. V., et al., (1999) NatureBiotechnol.,17:969-973; Terskikh et al., Science (2000)290:1585-8;Tsien, Annual Rev. of Biochemistry (1998) 67:509-544; Tsien,Nat. Biotech. (1999) 17:956-957; Ward et al., J. Biol. Chem. (1979)254:781-788; Wiedermann et al., Jarhrestagung der Deutschen Gesellschactfur Tropenokologie-gto. Ulm. 17-19.02.1999. Poster P4.20; Yarbrough etal., Proc Natl Acad Sci USA (2001) 98:462-7.

SUMMARY OF THE INVENTION

Nucleic acid compositions encoding novel chromo/fluoroproteins andmutants thereof, as well as the proteins encoded the same, are provided.The proteins of interest are proteins that are colored and/orfluorescent, where this feature arises from the interaction of two ormore residues of the protein. The subject proteins are furthercharacterized in that they are either obtained from non-bioluminescentCnidarian, e.g., Anthozoan, species or are obtained from Anthozoannon-Pennatulacean (sea pen) species. Specific proteins of interestinclude the following specific proteins: (1) Green fluorescent proteinfrom Heteractis crispa (hcriGFP); (2) Green fluorescent protein fromDendronephthya sp. (dendGFP); (3) Red fluorescent protein from Zoanthussp. (zoanRFP); (4) Green fluorescent protein from Scolymia cubensis(scubGFP1); (5) Green fluorescent protein from Scolymia cubensis(scubGFP2); (6) Red fluorescent protein from Ricordea florida (rfloRFP);(7) Green fluorescent protein from Ricordea florida (rfloGFP); (8) Redfluorescent protein from Montastraea cavernosa (mcavRFP); (9) Greenfluorescent protein from Montastraea cavernosa (mcavGFP); (10) Greenfluorescent protein from Condylactis gigantea (cgigGFP); (11) Greenfluorescent protein from Agaricia fragilis (afraGFP); (12) Greenfluorescent protein from Ricordea florida (rfloGFP2); (13) Greenfluorescent protein from Montastraea cavernosa (mcavGFP2); and (14)Green fluorescent protein homolog from Montastraea annularis (mannFP).Also of interest are proteins that are-substantially similar to, ormutants of, the above specific proteins. Also provided are fragments ofthe nucleic acids and the peptides encoded thereby, as well asantibodies to the subject proteins and transgenic cells and organisms.The subject protein and nucleic acid compositions find use in a varietyof different applications. Finally, kits for use in such applications,e.g., that include the subject nucleic acid compositions, are provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Changes of emission spectra during maturation of the newred-emitters: zoan2RFP (A, B), mcavRFP (C, D) and rfloRFP (E, F). Theexcitation wavelength is given within each graph. Horizontal axis iswavelength in nanometers, vertical axis is fluorescence intensity.Maturation stages: A, C, E—early; B, D, F—late (see Methods fordetails). All the three proteins exhibit “timer” phenotype (greenemission at first and red emission arising later). Note that zoan2RFPmatures significantly faster than mcavRFP and rfloRFP: even at the“early” stage the red emission peak is very pronounced, and by the“late” stage the protein converts into red completely. In contrast,mcavRFP and rfloRFP fail to undergo such a complete maturation.

FIG. 2. Details on excitation spectra of mcavRFP (A, B) and rfloRFP (C,D). Wavelengths at which the emission was monitored are given within thegraphs. A, C: excitation spectra of the green emission band in theimmature protein, lacking the red emission; B, D: excitation spectra ofthe red emission band in more mature form. Horizontal axis is wavelengthin nanometers, vertical axis is fluorescence intensity. Note that inboth proteins, the major excitation peaks for immature green and maturered forms are virtually identical to each other.

FIG. 3. The maximum-likelihood phylogenetic tree for the current datasetof anthozoan GFP-like proteins. Numbers at nodes denote thequartet-puzzling support values (1000 puzzling attempts). Proteins fromAlcyonaria sub-class, which were considered outgroups, are labeled inwhite on black. The “stem” of the tree (thick gray line), joining tworooting groups, putatively reflects the diversity of GFP-like proteinsbefore the separation of Alcyonaria and Zoantharia sub-classes. Graybars marked A, B, C and D denote four distinct clades of GFP-likeproteins found in Zoantharia. Scale bar: 0.1 replacements/site.

FIG. 4. Summary of spectral features and chromophore structures in thefamily of GFP-like proteins. Note that this paper uses different namesfor GFP-like proteins than proposed in original publications (theoriginal names, where available, are given in brackets in the firstcolumn; see text for nomenclature details).

FIG. 5. Summary of the major clades of GFP-like proteins from sub-classZoantharia.

FIG. 6. Excitation (solid lines) and emission (dotted lines) spectra forthe GFP-like proteins reported in this paper. The wavelengths at whichthe excitation or emission curves were taken are given in the legend toeach graph. Horizontal axis is wavelength in nanometers, vertical axisis fluorescence intensity. The graphs for the two new orange-redproteins are boxed.

FIG. 7. Alignment of the currently cloned and spectroscopicallycharacterized GFP-like proteins. Numeration above the alignment isaccording to GFP from Aequorea victoria.

FIG. 8 provides the nucleotide and amino acid sequence of wild typeHeteractis crispa hcriGFP. (SEQ ID NO:01 & 02)

FIG. 9 provides the nucleotide and amino acid sequence of wild typeDendronephthya sp. dendGFP. (SEQ ID NO:03 & 04)

FIG. 10 provides the nucleotide and amino acid sequence of wild typeZoanthus sp. zoanRFP. (SEQ ID NO:05 & 06)

FIG. 11 provides the nucleotide and amino acid sequence of wild typeScolymia cubensis scubGFP1. (SEQ ID NO:07 & 08)

FIG. 12 provides the nucleotide and amino acid sequence of wild typeScolymia cubensis scubGFP2. (SEQ ID NO:09 & 10)

FIG. 13 provides the nucleotide and amino acid sequence of wild typeRicordea florida rfloRFP. (SEQ ID NO:11 & 12)

FIG. 14 provides the nucleotide and amino acid sequence of wild typeRicordea florida rfloGFP. (SEQ ID NO:13 & 14)

FIG. 15 provides the nucleotide and amino acid sequence of wild typeMontastraea cavernosa mcavRFP. (SEQ ID NO:15 & 16)

FIG. 16 provides the nucleotide and amino acid sequence of wild typeMontastraea cavernosa mcavGFP. (SEQ ID NO:17 & 18)

FIG. 17 provides the nucleotide and amino acid sequence of wild typeCondylactis gigantea cgigGFP. (SEQ ID NO:19 & 20).

FIG. 18 provides the nucleotide and amino acid sequence of wild typeAgaricia fragilis afraGFP. (SEQ ID NO: 21& 22).

FIG. 19 provides the nucleotide and amino acid sequence of wild typeRicordea florida rfloGFP2. (SEQ ID NO: 23& 24).

FIG. 20 provides the nucleotide and amino acid sequence of wild typeMontastraea cavernosa mcavGFP2. (SEQ ID NO: 25& 26).

FIG. 21 provides the nucleotide and amino acid sequence of wild typeMontastraea annularis mannFP. (SEQ ID NO: 27& 28).

FEATURES OF THE INVENTION

The subject invention provides a nucleic acid present in other than itsnatural environment, wherein the nucleic acid encodes a chromo- orfluorescent protein and is from a non-bioluminescent Cnidarian species.In certain embodiments, the non-bioluminescent Cnidarian species is anAnthozoanspecies. In certain embodiments, the nucleic acid is isolated.In certain embodiments, the nucleic acid is present in other than itsnatural environment, where the nucleic acid encodes an Anthozoan chromo-or fluorescent protein and is from a non-Pennatulacean Anthozoanspecies. In certain embodiments, the nucleic acid has a sequence ofresidues that is substantially the same as or identical to a nucleotidesequence of at least 10 residues in length of SEQ ID NOS:01, 03, 05, 07,09, 11, 13, 15, 17; 19; 21; 23; 25; and 27. In certain embodiments, thenucleic acid has a sequence similarity of at least about 60% with asequence of at least 10 residues in length selected from the group ofsequences consisting of SEQ ID NOS:01, 03, 05, 07, 09, 11, 13, 15, 17;19; 21; 23; 25; and 27. In certain embodiments, the nucleic acid encodesa chromo and/or fluorescent protein that is either: (a) from anon-bioluminescent Cnidarian species; or (b) from a non-PennatulaceanAnthozoan species. In certain embodiments, the nucleic acid encodes aprotein that has an amino acid sequence selected from the groupconsisting of: SEQ ID NOS: 02; 04; 06; 08; 10; 12; 14; 16; 18; 20; 22;24; 26; and 28. In certain embodiments, the nucleic acid encodes amutant protein of a chromo and/or fluorescent protein that is either:(a) from a non-bioluminescent Cnidarian species; or (b) from anon-Pennatulacean Anthozoan species; where in certain embodiments themutant protein comprises at least one point mutation as compared to itswild type protein; and in other embodiments the mutant protein comprisesat least one deletion mutation as compared to its wild type protein.

Also provided are fragments of the provided nucleic acids. Also providedare isolated nucleic acids or mimetics thereof that hybridize understringent conditions to the provided nucleic acids. Also provided areconstructs comprising a vector and a nucleic acid or the presentinvention. Also provided are expression cassettes that include: (a) atranscriptional initiation region functional in an expression host; (b)a nucleic acid of the present invention; and (c) a transcriptionaltermination region functional in said expression host. Also provided arecells, or the progeny thereof, comprising an expression cassette of thepresent invention as part of an extrachromosomal element or integratedinto the genome of a host cell as a result of introduction of saidexpression cassette into said host cell.

Also provided are methods of producing a chromo and/or fluorescentprotein that include: growing a cell of the present invention, wherebysaid protein is expressed; and isolating said protein substantially freeof other proteins.

Also provided are proteins or fragments thereof encoded by a nucleicacid of the present invention.

Also provided are antibodies binding specifically to a protein of thepresent invention.

Also provided are transgenic cells or the progeny thereof that include atransgene selected that includes a nucleic acid of the presentinvention.

Also provided are transgenic organisms that include a transgene thatincludes a nucleic acid of the present invention.

Also provided are applications that employ a chromo- or fluorescentprotein of the present invention.

Also provided are applications that employ a nucleic acid encoding achromo- or fluorescent protein of the present invention.

Also provided are kits that include a nucleic acid according the subjectinvention and instructions for using said nucleic acid.

Definitions

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Maniatis, Fritsch & Sambrook,“Molecular Cloning: A Laboratory Manual (1982); “DNA Cloning: APractical Approach,” Volumes I and II (D. N. Glover ed. 1985);“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” (B. D. Hames & S. J. Higgins eds. (1985)); “Transcriptionand Translation” (B. D. Hames & S. J. Higgins eds. (1984)); “Animal CellCulture” (R. I. Freshney, ed. (1986)); “Immobilized Cells and Enzymes”(IRL Press, (1986)); B. Perbal, “A Practical Guide To Molecular Cloning”(1984).

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in either single stranded formor a double-stranded helix. This term refers only to the primary andsecondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes.

A DNA “coding sequence” is a DNA sequence which is transcribed andtranslated into a polypeptide in vivo when placed under the control ofappropriate regulatory sequences. The boundaries of the coding sequenceare determined by a start codon at the 5′ (amino) terminus and atranslation stop codon at the 3′ (carboxyl) terminus. A coding sequencecan include, but is not limited to, prokaryotic sequences, cDNA fromeukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian)DNA, and synthetic DNA sequences. A polyadenylation signal andtranscription termination sequence may be located 3′ to the codingsequence.

As used herein, the term “hybridization” refers to the process ofassociation of two nucleic acid strands to form an antiparallel duplexstabilized by means of hydrogen bonding between residues of the oppositenucleic acid strands.

The term “oligonucleotide” refers to a short (under 100 bases in length)nucleic acid molecule.

“DNA regulatory sequences”, as used herein, are transcriptional andtranslational control sequences, such as promoters, enhancers,polyadenylation signals, terminators, and the like, that provide forand/or regulate expression of a coding sequence in a host cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site, as well asprotein binding domains responsible for the binding of is RNApolymerase. Eukaryotic promoters will often, but not always, contain“TATA” boxes and “CAT” boxes. Various promoters, including induciblepromoters, may be used to drive the various vectors of the presentinvention.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A cell has been “transformed” or “transfected” by exogenous orheterologous DNA when such DNA has been introduced inside the cell. Thetransforming DNA may or may not be integrated (covalently linked) intothe genome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. In another example, heterologous DNA includes coding sequencein a construct where portions of genes from two different sources havebeen brought together so as to produce a fusion protein product. Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA as defined herein.

As used herein, the term “reporter gene” refers to a coding sequenceattached to heterologous promoter or enhancer elements and whose productmay be assayed easily and quantifiably when the construct is introducedinto tissues or cells.

The amino acids described herein are preferred to be in the “L” isomericform. The amino acid sequences are given in one-letter code (A: alanine;C: cysteine; D: aspartic acid; E: glutamic acid; F: phenylalanine; G:glycine; H: histidine; 1: isoleucine; K: lysine; L: leucine; M:methionine; N: asparagine; P: proline; Q: glutamine; R: arginine; S:serine; T: threonine; V: valine; W: tryptophan; Y: tyrosine; X: anyresidue). NH₂ refers to the free amino group present at the aminoterminus of a polypeptide. COOH refers to the free carboxy group presentat the carboxy terminus of a polypeptide. In keeping with standardpolypeptide nomenclature, J. Biol. Chem., 243 (1969), 3552-59 is used.

The term “immunologically active” defines the capability of the natural,recombinant or synthetic chromo/fluorescent protein, or any oligopeptidethereof, to induce a specific immune response in appropriate animals orcells and to bind with specific antibodies. As used herein, “antigenicamino acid sequence” means an amino acid sequence that, either alone orin association with a carrier molecule, can elicit an antibody responsein a mammal. The term “specific binding,” in the context of antibodybinding to an antigen, is a term well understood in the art and refersto binding of an antibody to the antigen to which the antibody wasraised, but not other, unrelated antigens.

As used herein the term “isolated” is meant to describe apolynucleotide, a polypeptide, an antibody, or a host cell that is in anenvironment different from that in which the polynucleotide, thepolypeptide, the antibody, or the host cell naturally occurs.

Bioluminescence (BL) is defined as emission of light by living organismsthat is well visible in the dark and affects visual behavior of animals(See e.g., Harvey, E. N. (1952). Bioluminescence. New York: AcademicPress; Hastings, J. W. (1995). Bioluminescence. In: Cell Physiology (ed.by N. Speralakis). pp. 651-681. New York: Academic Press.; Wilson, T.and Hastings, J. W. (1998). Bioluminescence. Annu Rev Cell Dev Biol 14,197-230.). Bioluminescence does not include so-called ultra-weak lightemission, which can be detected in virtually all living structures usingsensitive luminometric equipment (Murphy, M. E. and Sies, H.(1990).Visible-range low-level chemiluminescence in biological systems.Meth.Enzymol.186, 595-610; Radotic, K, Radenovic, C, Jeremic, M. (1998.)Spontaneous ultra-weak bioluminescence in plants: origin, mechanisms andproperties. Gen Physiol Biophys 17, 289-308), and from weak lightemission which most probably does not play any ecological role, such asthe glowing of bamboo growth cone (Totsune, H., Nakano, M., Inaba,H.(1993). Chemiluminescence from bamboo shoot cut. Biochem. Biophys.ResComm. 194,1025-1029) or emission of light during fertilization of animaleggs (Klebanoff, S. J., Froeder, C. A., Eddy, E. M., Shapiro, B. M.(1979). Metabolic similarities between fertilization and phagocytosis.Conservation of peroxidatic mechanism. J. Exp. Med. 149, 938-953;Schomer, B. and Epel, D. (1998). Redox changes during fertilization andmaturation of marine invertebrate eggs. Dev Biol 203, 1-11).

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Nucleic acid compositions encoding novel chromo/fluoroproteins andmutants thereof, as well as the proteins encoded the same, are provided.The proteins of interest are proteins that are colored and/orfluorescent, where this feature arises from the interaction of two ormore residues of the protein. The subject proteins are furthercharacterized in that they are either obtained from non-bioluminescentCnidarian, e.g., Anthozoan, species or are obtained fromnon-Pennatulacean (sea pen) Anthozoan species. Specific proteins ofinterest include the following specific proteins: (1) Green fluorescentprotein from Heteractis crispa (hcriGFP); (2) Green fluorescent proteinfrom Dendronephthya sp. (dendGFP); (3) Red fluorescent protein fromZoanthus sp. (zoanRFP); (4) Green fluorescent protein from Scolymiacubensis (scubGFP1); (5) Green fluorescent protein from Scolymiacubensis (scubGFP2); (6) Red fluorescent protein from Ricordea florida(rfloRFP); (7) Green fluorescent protein from Ricordea florida(rfloGFP); (8) Red fluorescent protein from Montastraea cavernosa(mcavRFP); (9) Green fluorescent protein from Montastraea cavernosa(mcavGFP); (10) Green fluorescent protein from Condylactis gigantea(cgigGFP); (11) Green fluorescent protein from Agaricia fragilis(afraGFP); (12) Green fluorescent protein from Ricordea florida(rfloGFP2); (13) Green fluorescent protein from Montastraea cavernosa(mcavGFP2); and (14) Green fluorescent protein homolog from Montastraeaannularis (mannFP). Also of interest are proteins that are substantiallysimilar to, or mutants of, the above specific proteins. Also providedare fragments of the nucleic acids and the peptides encoded thereby, aswell as antibodies to the subject proteins, and transgenic cells andorganisms that include the subject nucleic acid/protein compositions.The subject protein and nucleic acid compositions find use in a varietyof different applications. Finally, kits for use in such applications,e.g., that include the subject nucleic acid compositions, are provided.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, the singular forms “a,”“an” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing the cell lines, vectors,methodologies and other invention components that are described in thepublications which might be used in connection with the presentlydescribed invention.

In further describing the subject invention, the subject nucleic acidcompositions will be described first, followed by a discussion of thesubject protein compositions, antibody compositions and transgeniccells/organisms. Next a review of representative methods in which thesubject proteins find use is provided.

Nucleic Acid Compositions

As summarized above, the subject invention provides nucleic acidcompositions encoding chromo- and fluoroproteins and mutants thereof, aswell as fragments and homologues of these proteins. By chromo and/orfluorescent protein is meant a protein that is colored, i.e., ispigmented, where the protein may or may not be fluorescent, e.g., it mayexhibit low, medium or high fluorescence upon irradiation with light ofan excitation wavelength. In any event, the subject proteins of interestare those in which the colored characteristic, i.e., the chromo and/orfluorescent characteristic, is one that arises from the interaction oftwo or more residues of the protein, and not from a single residue, morespecifically a single side chain of a single residue, of the protein. Assuch, fluorescent proteins of the subject invention do not includeproteins that exhibit fluorescence only from residues that act bythemselves as intrinsic fluors, i.e., tryptophan, tyrosine andphenylalanine. As such, the fluorescent proteins of the subjectinvention are fluorescent proteins whose fluorescence arises from somestructure in the protein that is other than the above specified singleresidues, e.g., it arises from an interaction of two or more residues.

By nucleic acid composition is meant a composition comprising a sequenceof DNA having an open reading frame that encodes a chromo/fluoropolypeptide of the subject invention, i.e., a chromo/fluoroprotein gene,and is capable, under appropriate conditions, of being expressed as achromo/fluoro protein according to the subject invention. Alsoencompassed in this term are nucleic acids that are homologous,substantially similar or identical to the nucleic acids of the presentinvention. Thus, the subject invention provides genes and codingsequences thereof encoding the proteins of the subject invention, aswell as homologs thereof. The subject nucleic acids are present in otherthan their natural environment, e.g., they are isolated, present inenriched amounts, etc., from their naturally occurring environment,e.g., the organism from which they are obtained.

The nucleic acids are further characterized in that they encode proteinsthat are either from: (1) non-bioluminescent species, oftennon-bioluminescent Cnidarian species, e.g., non-bioluminescent Anthozoanspecies; or (2) from Anthozoan species that are not Pennatulaceanspecies, i.e., that are not sea pens. As such, the nucleic acids mayencode proteins from bioluminescent Anthozoan species, so long as thesespecies are not Pennatulacean species, e.g., that are not Renillan orPtilosarcan species. Specific nucleic acids of interest are those thatencode the following specific proteins: (1) Green fluorescent proteinfrom Heteractis crispa (hcriGFP) (Genbank Accession No. AF420592); (2)Green fluorescent protein from Dendronephthya sp. (dendGFP) (GenbankAccession No. AF420591); (3) Red fluorescent protein from Zoanthus sp.(zoanRFP) (Genbank Accession No. AY059642); (4) Green fluorescentprotein from Scolymia cubensis (scubGFP1) (Genbank Accession No.AY037767); (5) Green fluorescent protein from Scolymia cubensis(scubGFP2) (Genbank Accession No. AY037771); (6) Red fluorescent proteinfrom Ricordea florida (rfloRFP) (Genbank Accession No. AY037773); (7)Green fluorescent protein from Ricordea florida (rfloGFP) (GenbankAccession No. AY037772); (8) Red fluorescent protein from Montastraeacavernosa (mcavRFP) (Genbank Accession No. AY037770); (9) Greenfluorescent protein from Montastraea cavernosa (mcavGFP) (GenbankAccession No. AY037769); (10) Green fluorescent protein from Condylactisgigantea (cgigGFP) (Genbank Accession No. AY03776); (11) Greenfluorescent protein from Agaricia fragilis (afraGFP); (12) Greenfluorescent protein from Ricordea florida (rfloGFP2); (13) Greenfluorescent protein from Montastraea cavernosa (mcavGFP2); and (14)Green fluorescent protein homolog from Montastraea annularis (mannFP).Also of interest are derived from, or are mutants, homologues of, theabove specific nucleic acids.

In addition to the above described specific nucleic acid compositions,also of interest are homologues of the above sequences. With respect tohomologues of the subject nucleic acids, the source of homologous genesmay be any species of plant or animal or the sequence may be wholly orpartially synthetic. In certain embodiments, sequence similarity betweenhomologues is at least about 20%, sometimes at least about 25%, and maybe 30%, 35%, 40%, 50%, 60%, 70% or higher, including 75%, 80%, 85%, 90%and 95% or higher. Sequence similarity is calculated based on areference sequence, which may be a subset of a larger sequence, such asa conserved motif, coding region, flanking region, etc. A referencesequence will usually be at least about 18 nt long, more usually atleast about 30 nt long, and may extend to the complete sequence that isbeing compared. Algorithms for sequence analysis are known in the art,such as BLAST, described in Altschul et al. (1990), J. Mol. Biol.215:403-10 (using default settings, i.e. parameters w=4 and T=17). Thesequences provided herein are essential for recognizing related andhomologous nucleic acids in database searches. Of particular interest incertain embodiments are nucleic acids of substantially the same lengthas the nucleic acid identified as SEQ ID NOS: 01, 03, 05, 07, 09, 11,13, 15, 17, 19, 21, 23, 25 or 27, where by substantially the same lengthis meant that any difference in length does not exceed about 20 number%, usually does not exceed about 10 number % and more usually does notexceed about 5 number %; and have sequence identity to any of thesesequences of at least about 90%, usually at least about 95% and moreusually at least about 99% over the entire length of the nucleic acid.In many embodiments, the nucleic acids have a sequence that issubstantially similar (i.e. the same as) or identical to the sequencesof SEQ ID NOS: 01, 03, 05, 07, 09, 11, 13, 15, 17, 21, 23, 25, 27. Bysubstantially similar is meant that sequence identity will generally beat least about 60%, usually at least about 75% and often at least about80, 85, 90, or even 95%.

Also provided are nucleic acids that encode the proteins encoded by theabove described nucleic acids, but differ in sequence from the abovedescribed nucleic acids due to the degeneracy of the genetic code.

Also provided are nucleic acids that hybridize to the above describednucleic acid under stringent conditions. An example of stringenthybridization conditions is hybridization at 50° C. or higher and0.1×SSC (15 mM sodium chloride/1.5 mM sodium citrate). Another exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA,followed by washing the filters in 0.1×SSC at about 65° C. Stringenthybridization conditions are hybridization conditions that are at leastas stringent as the above representative conditions, where conditionsare considered to be at least as stringent if they are at least about80% as stringent, typically at least about 90% as stringent as the abovespecific stringent conditions. Other stringent hybridization conditionsare known in the art and may also be employed to identify nucleic acidsof this particular embodiment of the invention.

Nucleic acids encoding mutants of the proteins of the invention are alsoprovided. Mutant nucleic acids can be generated by random mutagenesis ortargeted mutagenesis, using well-known techniques which are routine inthe art. In some embodiments, chromo- or fluorescent proteins encoded bynucleic acids encoding homologues or mutants have the same fluorescentproperties as the wild-type fluorescent protein. In other embodiments,homologue or mutant nucleic acids encode chromo- or fluorescent proteinswith altered spectral properties, as described in more detail herein.

One category of mutant that is of particular interest is thenon-aggregating mutant. In many embodiments, the non-aggregating mutantdiffers from the wild type sequence by a mutation in the N-terminus thatmodulates the charges appearing on side groups of the N-terminusresidues, e.g., to reverse or neutralize the charge, in a mannersufficient to produce a non-aggregating mutant of the naturallyoccurring protein or mutant, where a particular protein is considered tobe non-aggregating if it is determined be non-aggregating using theassay reported in U.S. Patent Application Ser. No. 60/270,983, thedisclosure of which is herein incorporated by reference.

Another category of mutant of particular interest is the modulatedoligomerization mutant. A mutant is considered to be a modulatedoligomerization mutant if its oligomerization properties are differentas compared to the wild type protein. For example, if a particularmutant oligomerizes to a greater or lesser extent than the wild type, itis considered to be an oligomerization mutant. Of particular interestare oligomerization mutants that do not oligomerize, i.e., are monomersunder physiological (e.g., intracellular) conditions, or oligomerize toa lesser extent that the wild type, e.g., are dimers or trimers underintracellular conditions.

Nucleic acids of the subject invention may be cDNA or genomic DNA or afragment thereof. In certain embodiments, the nucleic acids of thesubject invention include one or more of the open reading framesencoding specific fluorescent proteins and polypeptides, and introns, aswell as adjacent 5′ and 3′ non-coding nucleotide sequences involved inthe regulation of expression, up to about 20 kb beyond the codingregion, but possibly further in either direction. The subject nucleicacids may be introduced into an appropriate vector for extrachromosomalmaintenance or for integration into a host genome, as described ingreater detail below.

The term “cDNA” as used herein is intended to include all nucleic acidsthat share the arrangement of sequence elements found in native maturemRNA species, where sequence elements are exons and 5′ and 3′ non-codingregions. Normally mRNA species have contiguous exons, with theintervening introns, when present, being removed by nuclear RNAsplicing, to create a continuous open reading frame encoding theprotein.

A genomic sequence of interest comprises the nucleic acid presentbetween the initiation codon and the stop codon, as defined in thelisted sequences, including all of the introns that are normally presentin a native chromosome. It may further include 5′ and 3′ un-translatedregions found in the mature mRNA. It may further include specifictranscriptional and translational regulatory sequences, such aspromoters, enhancers, etc., including about 1 kb, but possibly more, offlanking genomic DNA at either the 5′ or 3′ end of the transcribedregion. The genomic DNA may be isolated as a fragment of 100 kbp orsmaller; and substantially free of flanking chromosomal sequence. Thegenomic DNA flanking the coding region, either 3′ or 5′, or internalregulatory sequences as sometimes found in introns, contains sequencesrequired for proper tissue and stage specific expression.

The nucleic acid compositions of the subject invention may encode all ora part of the subject proteins. Double or single stranded fragments maybe obtained from the DNA sequence by chemically synthesizingoligonucleotides in accordance with conventional methods, by restrictionenzyme digestion, by PCR amplification, etc. For the most part, DNAfragments will be of at least about 15 nt, usually at least about 18 ntor about 25 nt, and may be at least about 50 nt. In some embodiments,the subject nucleic acid molecules may be about 100 nt, about 200 nt,about 300 nt, about 400 nt, about 500 nt, about 600 nt, about 700 nt, orabout 720 nt in length. The subject nucleic acids may encode fragmentsof the subject proteins or the full-length proteins, e.g., the subjectnucleic acids may encode polypeptides of about 25 aa, about 50 aa, about75 aa, about 100 aa, about 125 aa, about 150 aa, about 200 aa, about 210aa, about 220 aa, about 230 aa, or about 240 aa, up to the entireprotein.

The subject nucleic acids are isolated and obtained in substantialpurity, generally as other than an intact chromosome. Usually, the DNAwill be obtained substantially free of other nucleic acid sequences thatdo not include a nucleic acid of the subject invention or fragmentthereof, generally being at least about 50%, usually at least about 90%pure and are typically “recombinant”, i.e. flanked by one or morenucleotides with which it is not normally associated on a naturallyoccurring chromosome.

The subject polynucleotides (e.g., a polynucleotide having a sequence ofSEQ ID NOS: 01, 03, 05, 07, 09, 11, 13, 15, 17, 19, 21, 23, 25, 27etc.), the corresponding cDNA, the full-length gene and constructs ofthe subject polynucleotides are provided. These molecules can begenerated synthetically by a number of different protocols known tothose of skill in the art. Appropriate polynucleotide constructs arepurified using standard recombinant DNA techniques as described in, forexample, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., (1989) Cold Spring Harbor Press, Cold Spring Harbor, N.Y., andunder current regulations described in United States Dept. of HHS,National Institute of Health (NIH) Guidelines for Recombinant DNAResearch.

Also provided are nucleic acids that encode fusion proteins of thesubject proteins, or fragments thereof, which are fused to a secondprotein, e.g., a degradation sequence, a signal peptide, etc. Fusionproteins may comprise a subject polypeptide, or fragment thereof, and anon-Anthozoan polypeptide (“the fusion partner”) fused in-frame at theN-terminus and/or C-terminus of the subject polypeptide. Fusion partnersinclude, but are not limited to, polypeptides that can bind antibodyspecific to the fusion partner (e.g., epitope tags); antibodies orbinding fragments thereof; polypeptides that provide a catalyticfunction or induce a cellular response; ligands or receptors or mimeticsthereof; and the like. In such fusion proteins, the fusion partner isgenerally not naturally associated with the subject Anthozoan portion ofthe fusion protein, and is typically not an Anthozoan protein orderivative/fragment thereof, i.e., it is not found in Anthozoan species.

Also provided are constructs comprising the subject nucleic acidsinserted into a vector, where such constructs may be used for a numberof different applications, including propagation, protein production,etc. Viral and non-viral vectors may be prepared and used; includingplasmids. The choice of vector will depend on the type of cell in whichpropagation is desired and the purpose of propagation. Certain vectorsare useful for amplifying and making large amounts of the desired DNAsequence. Other vectors are suitable for expression in cells in culture.Still other vectors are suitable for transfer and expression in cells ina whole animal or person. The choice of appropriate vector is wellwithin the skill of the art. Many such vectors are availablecommercially. To prepare the constructs, the partial or full-lengthpolynucleotide is inserted into a vector typically by means of DNAligase attachment to a cleaved restriction enzyme site in the vector.Alternatively, the desired nucleotide sequence can be inserted byhomologous recombination in vivo. Typically this is accomplished byattaching regions of homology to the vector on the flanks of the desirednucleotide sequence. Regions of homology are added by ligation ofoligonucleotides, or by polymerase chain reaction using primerscomprising both the region of homology and a portion of the desirednucleotide sequence, for example.

Also provided are expression cassettes or systems that find use in,among other applications, the synthesis of the subject proteins. Forexpression, the gene product encoded by a polynucleotide of theinvention is expressed in any convenient expression system, including,for example, bacterial, yeast, insect, amphibian and mammalian systems.Suitable vectors and host cells are described in U.S. Pat. No.5,654,173. In the expression vector, a subject polynucleotide, e.g., asset forth in SEQ ID NOS:01; 03; 05; 07; 09; 11; 13; 15; 17; 19; 21; 23;25 or 27, is linked to a regulatory sequence as appropriate to obtainthe desired expression properties. These regulatory sequences caninclude promoters (attached either at the 5′ end of the sense strand orat the 3′ end of the antisense strand), enhancers, terminators,operators, repressors, and inducers. The promoters can be regulated orconstitutive. In some situations it may be desirable to useconditionally active promoters, such as tissue-specific or developmentalstage-specific promoters. These are linked to the desired nucleotidesequence using the techniques described above for linkage to vectors.Any techniques known in the art can be used. In other words, theexpression vector will provide a transcriptional and translationalinitiation region, which may be inducible or constitutive, where thecoding region is operably linked under the transcriptional control ofthe transcriptional initiation region, and a transcriptional andtranslational termination region. These control regions may be native tothe subject species from which the subject nucleic acid is obtained, ormay be derived from exogenous sources.

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present. Expression vectors may be usedfor, among other things, the production of fusion proteins, as describedabove.

Expression cassettes may be prepared comprising a transcriptioninitiation region, the gene or fragment thereof, and a transcriptionaltermination region. Of particular interest is the use of sequences thatallow for the expression of functional epitopes or domains, usually atleast about 8 amino acids in length, more usually at least about 15amino acids in length, to about 25 amino acids, and up to the completeopen reading frame of the gene. After introduction of the DNA, the cellscontaining the construct may be selected by means of a selectablemarker, the cells expanded and then used for expression.

The above described expression systems may be employed with prokaryotesor eukaryotes in accordance with conventional ways, depending upon thepurpose for expression. For large scale production of the protein, aunicellular organism, such as E. coli, B. subtilis, S. cerevisiae,insect cells in combination with baculovirus vectors, or cells of ahigher organism such as vertebrates, e.g. COS 7 cells, HEK 293, CHO,Xenopus Oocytes, etc., may be used as the expression host cells. In somesituations, it is desirable to express the gene in eukaryotic cells,where the expressed protein will benefit from native folding andpost-translational modifications. Small peptides can also be synthesizedin the laboratory. Polypeptides that are subsets of the complete proteinsequence may be used to identify and investigate parts of the proteinimportant for function.

Specific expression systems of interest include bacterial, yeast, insectcell and mammalian cell derived expression systems. Representativesystems from each of these categories is are provided below:

Bacteria. Expression systems in bacteria include those described inChang et al., Nature (1978) 275:615; Goeddel et al., Nature (1979)281:544; Goeddel et al., Nucleic Acids Res. (1980) 8:4057; EP 0 036,776;U.S. Pat. No. 4,551,433; DeBoer et al., Proc. Natl. Acad. Sci. (USA)(1983) 80:21-25; and Siebenlist et al., Cell (1980) 20:269.

Yeast. Expression systems in yeast include those described in Hinnen etal., Proc. Natl. Acad. Sci. (USA) (1978) 75:1929; Ito et al., J.Bacteriol. (1983) 153:163; Kurtz et al., Mol. Cell. Biol. (1986) 6:142;Kunze et al., J. Basic Microbiol. (1985) 25:141; Gleeson et al., J. Gen.Microbiol. (1986) 132:3459; Roggenkamp et al., Mol. Gen. Genet. (1986)202:302; Das et al., J. Bacteriol. (1984) 158:1165; De Louvencourt etal., J. Bacteriol. (1983) 154:737; Van den Berg et al., Bio/Technology(1990) 8:135; Kunze et al., J. Basic Microbiol. (1985) 25:141; Cregg etal., Mol. Cell. Biol. (1985) 5:3376; U.S. Pat. Nos. 4,837,148 and4,929,555; Beach and Nurse, Nature (1981) 300:706; Davidow et al., Curr.Genet. (1985) 10:380; Gaillardin et al., Curr. Genet. (1985) 10:49;Ballance et al., Biochem. Biophys. Res. Commun. (1983) 112:284-289;Tilburn et al., Gene (1983) 26:205-221; Yelton et al., Proc. Natl. Acad.Sci. (USA) (1984) 81:1470-1474; Kelly and Hynes, EMBO J. (1985)4:475479; EP 0 244,234; and WO 91/00357.

Insect Cells. Expression of heterologous genes in insects isaccomplished as described in U.S. Pat. No. 4,745,051; Friesen et al.,“The Regulation of Baculovirus Gene Expression”, in: The MolecularBiology Of Baculoviruses (1986) (W. Doerfler, ed.); EP 0 127,839; EP 0155,476; and Vlak et al., J. Gen. Virol. (1988) 69:765-776; Miller etal., Ann. Rev. Microbiol. (1988) 42:177; Carbonell et al., Gene (1988)73:409; Maeda et al., Nature (1985) 315:592-594; Lebacq-Verheyden etal., Mol. Cell. Biol. (1988) 8:3129; Smith et al., Proc. Natl. Acad.Sci. (USA) (1985) 82:8844; Miyajima et al., Gene (1987) 58:273; andMartin et al., DNA (1988) 7:99. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts aredescribed in Luckow et al., Bio/Technology (1988) 6:47-55, Miller etal., Generic Engineering (1986) 8:277-279, and Maeda et al., Nature(1985) 315:592-594.

Mammalian Cells. Mammalian expression is accomplished as described inDijkema et al., EMBO J. (1985) 4:761, Gorman et al., Proc. Natl. Acad.Sci. (USA) (1982) 79:6777, Boshart et al., Cell (1985) 41:521 and U.S.Pat. No. 4,399,216. Other features of mammalian expression arefacilitated as described in Ham and Wallace, Meth. Enz. (1979) 58:44,Barnes and Sato, Anal. Biochem. (1980) 102:255, U.S. Pat. Nos.4,767,704, 4,657,866, 4,927,762, 4,560,655, WO 90/103430, WO 87/00195,and U.S. RE 30,985.

When any of the above host cells, or other appropriate host cells ororganisms, are used to replicate and/or express the polynucleotides ornucleic acids of the invention, the resulting replicated nucleic acid,RNA, expressed protein or polypeptide, is within the scope of theinvention as a product of the host cell or organism. The product isrecovered by any appropriate means known in the art.

Once the gene corresponding to a selected polynucleotide is identified,its expression can be regulated in the cell to which the gene is native.For example, an endogenous gene of a cell can be regulated by anexogenous regulatory sequence inserted into the genome of the cell atlocation sufficient to at least enhance expressed of the gene in thecell. The regulatory sequence may be designed to integrate into thegenome via homologous recombination, as disclosed in U.S. Pat. Nos.5,641,670 and 5,733,761, the disclosures of which are hereinincorporated by reference, or may be designed to integrate into thegenome via non-homologous recombination, as described in WO 99/15650,the disclosure of which is herein incorporated by reference. As such,also encompassed in the subject invention is the production of thesubject proteins without manipulation of the encoding nucleic aciditself, but instead through integration of a regulatory sequence intothe genome of cell that already includes a gene encoding the desiredprotein, as described in the above incorporated patent documents.

Also provided are homologs of the subject nucleic acids. Homologs areidentified by any of a number of methods. A fragment of the providedcDNA may be used as a hybridization probe against a cDNA library fromthe target organism of interest, where low stringency conditions areused. The probe may be a large fragment, or one or more short degenerateprimers. Nucleic acids having sequence similarity are detected byhybridization under low stringency conditions, for example, at 50° C.and 6×SSC (0.9 M sodium chloride/0.09 M sodium citrate) and remain boundwhen subjected to washing at 55° C. in 1×SSC (0.15 M sodiumchloride/0.015 M sodium citrate). Sequence identity may be determined byhybridization under stringent conditions, for example, at 50° C. orhigher and 0.1×SSC (15 mM sodium chloride/1.5 mM sodium citrate).Nucleic acids having a region of substantial identity to the providedsequences, e.g. allelic variants, genetically altered versions of thegene, etc., bind to the provided sequences under stringent hybridizationconditions. By using probes, particularly labeled probes of DNAsequences, one can isolate homologous or related genes.

Also of interest are promoter elements of the subject genomic sequences,where the sequence of the 5′ flanking region may be utilized forpromoter elements, including enhancer binding sites, e.g., that providefor regulation of expression in cells/tissues where the subject proteinsgene are expressed.

Also provided are small DNA fragments of the subject nucleic acids,which fragments are useful as primers for PCR, hybridization screeningprobes, etc. Larger DNA fragments, i.e., greater than 100 nt are usefulfor production of the encoded polypeptide, as described in the previoussection. For use in geometric amplification reactions, such as geometricPCR, a pair of primers will be used. The exact composition of the primersequences is not critical to the invention, but for most applicationsthe primers will hybridize to the subject sequence under stringentconditions, as known in the art. It is preferable to choose a pair ofprimers that will generate an amplification product of at least about 50nt, preferably at least about 100 nt. Algorithms for the selection ofprimer sequences are generally known, and are available in commercialsoftware packages. Amplification primers hybridize to complementarystrands of DNA, and will prime towards each other.

The DNA may also be used to identify expression of the gene in abiological specimen. The manner in which one probes cells for thepresence of particular nucleotide sequences, as genomic DNA or RNA, iswell established in the literature. Briefly, DNA or mRNA is isolatedfrom a cell sample. The mRNA may be amplified by RT-PCR, using reversetranscriptase to form a complementary DNA strand, followed by polymerasechain reaction amplification using primers specific for the subject DNAsequences. Alternatively, the mRNA sample is separated by gelelectrophoresis, transferred to a suitable support, e.g. nitrocellulose,nylon, etc., and then probed with a fragment of the subject DNA as aprobe. Other techniques, such as oligonucleotide ligation assays, insitu hybridizations, and hybridization to DNA probes arrayed on a solidchip may also find use. Detection of mRNA hybridizing to the subjectsequence is indicative of Anthozoan protein gene expression in thesample.

The subject nucleic acids, including flanking promoter regions andcoding regions, may be mutated in various ways known in the art togenerate targeted changes in promoter strength, sequence of the encodedprotein, properties of the encoded protein, including fluorescentproperties of the encoded protein, etc. The DNA sequence or proteinproduct of such a mutation will usually be substantially similar to thesequences provided herein, e.g. will differ by at least one nucleotideor amino acid, respectively, and may differ by at least two but not morethan about ten nucleotides or amino acids. The sequence changes may besubstitutions, insertions, deletions, or a combination thereof.Deletions may further include larger changes, such as deletions of adomain or exon, e.g. of stretches of 10, 20, 50, 75, 100,150 or more aaresidues. Techniques for in vitro mutagenesis of cloned genes are known.Examples of protocols for site specific mutagenesis may be found inGustin et al. (1993), Biotechniques 14:22; Barany (1985), Gene37:111-23; Colicelli et al. (1985), Mol. Gen. Genet 199:537-9; andPrentki et al. (1984), Gene 29:303-13. Methods for site specificmutagenesis can be found in Sambrook et al., Molecular Cloning: ALaboratory Manual, CSH Press 1989, pp. 15.3-15.108; Weiner et al.(1993), Gene 126:35-41; Sayers et al. (1992), Biotechniques 13:592-6;Jones and Winistorfer (1992), Biotechniques 12:528-30; Barton et al.(1990), Nucleic Acids Res 18:7349-55; Marotti and Tomich (1989), GeneAnal. Tech. 6:67-70; and Zhu (1989), Anal Biochem 177:120-4. Suchmutated nucleic acid derivatives may be used to study structure-functionrelationships of a particular chromo/fluorescent protein, or to alterproperties of the protein that affect its function or regulation.

Of particular interest in many embodiments is the following specificmutation protocol, which protocol finds use in mutating chromoproteins(e.g., colored proteins that have little if any fluorescence) intofluorescent mutants. In this protocol, the sequence of the candidateprotein is aligned with the amino acid sequence of Aequorea Victoriawild type GFP, according to the protocol reported in Matz et al.,“Fluorescent proteins from non-bioluminescent Anthozoa species,” NatureBiotechnology (October 1999) 17: 969-973. Residue 148 of the alignedchromoprotein is identified and then changed to Ser, e.g., by sitedirected mutagenesis, which results in the production of a fluorescentmutant of the wild type chromoprotein. See e.g., NFP-7 described below,which wild type protein is a chromoprotein that is mutated into afluorescent protein by substitution of Ser for the native Ala residue atposition 148.

Also of interest are humanized versions of the subject nucleic acids. Asused herein, the term “humanized” refers to changes made to the anucleic acid sequence to optimize the codons for expression of theprotein in human cells (Yang et al., Nucleic Acids Research 24 (1996),4592-4593). See also U.S. Pat. No. 5,795,737 which describeshumanization of proteins, the disclosure of which is herein incorporatedby reference.

Protein/Polypeptide Compositions

Also provided by the subject invention are chromo- and/or fluorescentproteins and mutants thereof, as well as polypeptide compositionsrelated thereto. As the subject proteins are chromoproteins, they arecolored proteins, which may be fluorescent, low or non-fluorescent. Asused herein, the terms chromoprotein and fluorescent protein do notinclude luciferases, such as Renilla luciferase, and refer to anyprotein that is pigmented or colored and/or fluoresces when irradiatedwith light, e.g., white light or light of a specific wavelength (ornarrow band of wavelengths such as an excitation wavelength). The termpolypeptide composition as used herein refers to both the full-lengthprotein, as well as portions or fragments thereof. Also included in thisterm are variations of the naturally occurring protein, where suchvariations are homologous or substantially similar to the naturallyoccurring protein, and mutants of the naturally occurring proteins, asdescribed in greater detail below. The subject polypeptides are presentin other than their natural environment.

In many embodiments, the subject proteins have an absorbance maximumranging from about 300 to 700, usually from about 350 to 650 and moreusually from about 400 to 600 nm. Where the subject proteins arefluorescent proteins, by which is meant that they can be excited at onewavelength of light following which they will emit light at anotherwavelength, the excitation spectra of the subject proteins typicallyranges from about 300 to 700, usually from about 350 to 650 and moreusually from about 400 to 600 nm while the emission spectra of thesubject proteins typically ranges from about 400 to 800, usually fromabout 425 to 775 and more usually from about 450 to 750 nm. The subjectproteins generally have a maximum extinction coefficient that rangesfrom about 10,000 to 50,000 and usually from about 15,000 to 45,000. Thesubject-proteins typically range in length from about 150 to 300 andusually from about 200 to 300 amino acid residues, and generally have amolecular weight ranging from about 15 to 35 kDa, usually from about17.5 to 32.5 kDa.

In certain embodiments, the subject proteins are bright, where by brightis meant that the chromoproteins and their fluorescent mutants can bedetected by common methods (e.g., visual screening, spectrophotometry,spectrofluorometry, fluorescent microscopy, by FACS machines, etc.)Fluorescence brightness of particular fluorescent proteins is determinedby its quantum yield multiplied by maximal extinction coefficient.Brightness of a chromoproteins may be expressed by its maximalextinction coefficient.

In certain embodiments, the subject proteins fold rapidly followingexpression in the host cell. By rapidly folding is meant that theproteins achieve their tertiary structure that gives rise to theirchromo- or fluorescent quality in a short period of time. In theseembodiments, the proteins fold in a period of time that generally doesnot exceed about 3 days, usually does not exceed about 2 days and moreusually does not exceed about 1 day.

Specific proteins of interest include the following specific proteins:(1) Green fluorescent protein from Heteractis crispa (hcriGFP); (2)Green fluorescent protein from Dendronephthya sp. (dendGFP); (3) Redfluorescent protein from Zoanthus sp. (zoanRFP); (4) Green fluorescentprotein from Scolymia cubensis (scubGFP1); (5) Green fluorescent proteinfrom Scolymia cubensis (scubGFP2); (6) Red fluorescent protein fromRicordea florida (rfloRFP); (7) Green fluorescent protein from Ricordeaflorida (rfloGFP); (8) Red fluorescent protein from Montastraeacavernosa (mcavRFP); (9) Green fluorescent protein from Montastraeacavernosa (mcavGFP); (10) Green fluorescent protein from Condylactisgigantea (cgigGFP); (11) Green fluorescent protein from Agariciafragilis (afraGFP); (12) Green fluorescent protein from Ricordea florida(rfloGFP2); (13) Green fluorescent protein from Montastraea cavernosa(mcavGFP2); and (14) Green fluorescent protein homolog from Montastraeaannularis (mannFP).

Homologs or proteins (or fragments thereof) that vary in sequence fromthe above provided specific amino acid sequences of the subjectinvention, i.e., SEQ ID NOS: 02; 04; 06; 08; 10; 12; 14; 16; 18; 20; 22;24; 26 or 28, are also provided. By homolog is meant a protein having atleast about 10%, usually at least about 20% and more usually at leastabout 30%, and in many embodiments at least about 35%, usually at leastabout 40% and more usually at least about 60% amino acid sequenceidentity to the protein of the subject invention, as determined usingMegAlign, DNAstar (1998) clustal algorithm as described in D. G. Higginsand P. M. Sharp, “Fast and Sensitive multiple Sequence Alignments on aMicrocomputer,” (1989) CABIOS, 5: 151-153. (Parameters used are ktuple1, gap penalty 3, window, 5 and diagonals saved 5). In many embodiments,homologues of interest have much higher sequence identify, e.g., 65%,70%, 75%, 80%, 85%, 90% or higher.

Also provided are proteins that are substantially identical to the wildtype protein, where by substantially identical is meant that the proteinhas an amino acid sequence identity to the sequence of wild type proteinof at least about 60%, usually at least about 65% and more usually atleast about 70%, where in some instances the identity may be muchhigher, e.g., 75%, 80%, 85%, 90%, 95% or higher.

In many embodiments, the subject homologues have structural featuresfound in the above provided specific sequences, where such structuralfeatures include the β-can fold.

Proteins which are mutants of the above-described naturally occurringproteins are also provided. Mutants may retain biological properties ofthe wild-type (e.g., naturally occurring) proteins, or may havebiological properties which differ from the wild-type proteins. The term“biological property” of the subject proteins includes, but is notlimited to, spectral properties, such as absorbance maximum, emissionmaximum, maximum extinction coefficient, brightness (e.g., as comparedto the wild-type protein or another reference protein such as greenfluorescent protein from A. Victoria), and the like; in vivo and/or invitro stability (e.g., half-life); etc. Mutants include single aminoacid changes, deletions of one or more amino acids, N-terminaltruncations, C-terminal truncations, insertions, etc.

Mutants can be generated using standard techniques of molecular biology,e.g., random mutagenesis, and targeted mutagenesis. Several mutants aredescribed herein. Given the guidance provided in the Examples, and usingstandard techniques, those skilled in the art can readily generate awide variety of additional mutants and test whether a biologicalproperty has been altered. For example, fluorescence intensity can bemeasured using a spectrophotometer at various excitation wavelengths.

Those proteins of the subject invention that are naturally occurringproteins are present in a non-naturally occurring environment, e.g., areseparated from their naturally occurring environment. In certainembodiments, the subject proteins are present in a composition that isenriched for the subject protein as compared to its naturally occurringenvironment. For example, purified protein is provided, where bypurified is meant that the protein is present in a composition that issubstantially free of non-chromo/fluoroprotein proteins of interest,where by substantially free is meant that less than 90%, usually lessthan 60% and more usually less than 50% of the composition is made up ofnon-chromoproteins or mutants thereof of interest. The proteins of thesubject invention may also be present as an isolate, by which is meantthat the protein is substantially free of other proteins and othernaturally occurring biologic molecules, such as oligosaccharides,polynucleotides and fragments thereof, and the like, where the term“substantially free” in this instance means that less than 70%, usuallyless than 60% and more usually less than 50% of the compositioncontaining the isolated protein is some other naturally occurringbiological molecule. In certain embodiments, the proteins are present insubstantially pure form, where by “substantially pure form” is meant atleast 95%, usually at least 97% and more usually at least 99% pure.

In addition to the naturally occurring proteins, polypeptides that varyfrom the naturally occurring proteins, e.g., the mutant proteinsdescribed above, are also provided. Generally such polypeptides includean amino acid sequence encoded by an open reading frame (ORF) of thegene encoding the subject wild type protein, including the full lengthprotein and fragments thereof, particularly biologically activefragments and/or fragments corresponding to functional domains, and thelike; and including fusions of the subject polypeptides to otherproteins or parts thereof. Fragments of interest will typically be atleast about 10 aa in length, usually at least about 50 aa in length, andmay-be as long as 300 aa in length or longer, but will usually notexceed about 1000 aa in length, where the fragment will have a stretchof amino acids that is identical to the subject protein of at leastabout 10 aa, and usually at least about 15 aa, and in many embodimentsat least about 50 aa in length. In some embodiments, the subjectpolypeptides are about 25 aa, about 50 aa, about 75 aa, about 100 aa,about 125 aa, about 150 aa, about 200 aa, about 210 aa, about 220 aa,about 230 aa, or about 240 aa in length, up to the entire protein. Insome embodiments, a protein fragment retains all or substantially all ofa biological property of the wild-type protein.

The subject proteins and polypeptides may be obtained from naturallyoccurring sources or synthetically produced. For example, wild typeproteins may be derived from biological sources which express theproteins, e.g., non-bioluminescent Cnidarian, e.g., Anthozoan, species,such as the specific ones listed above. The subject proteins may also bederived from synthetic means, e.g., by expressing a recombinant gene ornucleic acid coding sequence encoding the protein of interest in asuitable host, as described above. Any convenient protein purificationprocedures-may be employed, where suitable protein purificationmethodologies are described in Guide to Protein Purification, (Deuthsered.) (Academic Press, 1990). For example, a lysate may prepared from theoriginal source and purified using HPLC, exclusion chromatography, gelelectrophoresis, affinity chromatography, and the like.

Antibody Compositions

Also provided are antibodies that specifically bind to the subjectfluorescent proteins. Suitable antibodies are obtained by immunizing ahost animal with peptides comprising all or a portion of the subjectprotein. Suitable host animals include mouse, rat sheep, goat, hamster,rabbit, etc. The origin of the protein immunogen will generally be aCnidarian species, specifcally a non-bioluminescent Cnidarian species,such as an Anthozoan species or a non-Petalucean Anthozoan species. Thehost animal will generally be a different species than the immunogen,e.g., mice, etc.

The immunogen may comprise the complete protein, or fragments andderivatives thereof. Preferred immunogens comprise all or a part of theprotein, where these residues contain the post-translation modificationsfound on the native target protein. Immunogens are produced in a varietyof ways known in the art, e.g., expression of cloned genes usingconventional recombinant methods, isolation from Anthozoan species oforigin, etc.

For preparation of polyclonal antibodies, the first step is immunizationof the host animal with the target protein, where the target proteinwill preferably be in substantially pure form, comprising less thanabout 1% contaminant. The immunogen may comprise the complete targetprotein, fragments or derivatives thereof. To increase the immuneresponse of the host animal, the target protein may be combined with anadjuvant, where suitable adjuvants include alum, dextran, sulfate, largepolymeric anions, oil & water emulsions, e.g. Freund's adjuvant,Freund's complete adjuvant, and the like. The target protein may also beconjugated to synthetic carrier proteins or synthetic antigens. Avariety of hosts may be immunized to produce the polyclonal antibodies.Such hosts include rabbits, guinea pigs, rodents, e.g. mice, rats,sheep, goats, and the like. The target protein is administered to thehost, usually intradermally, with an initial dosage followed by one ormore, usually at least two, additional booster dosages. Followingimmunization, the blood from the host will be collected, followed byseparation of the serum from the blood cells. The Ig present in theresultant antiserum may be further fractionated using known methods,such as ammonium salt fractionation, DEAE chromatography, and the like.

Monoclonal antibodies are produced by conventional techniques.Generally, the spleen and/or lymph nodes of an immunized host animalprovide a source of plasma cells. The plasma cells are immortalized byfusion with myeloma cells to produce hybridoma cells. Culturesupernatant from individual hybridomas is screened using standardtechniques to identify those producing antibodies with the desiredspecificity. Suitable animals for production of monoclonal antibodies tothe human protein include mouse, rat, hamster, etc. To raise antibodiesagainst the mouse protein, the animal will generally be a hamster,guinea pig, rabbit, etc. The antibody may be purified from the hybridomacell supernatants or ascites fluid by conventional techniques, e.g.affinity chromatography using protein bound to an insoluble support,protein A sepharose, etc.

The antibody may be produced as a single chain, instead of the normalmultimeric structure. Single chain antibodies are described in Jost etal. (1994) J.B.C. 269:26267-73, and others. DNA sequences encoding thevariable region of the heavy chain and the variable region of the lightchain are ligated to a spacer encoding at least about 4 amino acids ofsmall neutral amino acids, including glycine and/or serine. The proteinencoded by this fusion allows assembly of a functional variable regionthat retains the specificity and affinity of the original antibody.

Also of interest in certain embodiments are humanized antibodies.Methods of humanizing antibodies are known in the art. The humanizedantibody may be the product of an animal having transgenic humanimmunoglobulin constant region genes (see for example InternationalPatent Applications WO 90/10077 and WO 90/04036). Alternatively, theantibody of interest may be engineered by recombinant DNA techniques tosubstitute the CH1, CH2, CH3, hinge domains, and/or the framework domainwith the corresponding human sequence (see WO 92/02190).

The use of Ig cDNA for construction of chimeric immunoglobulin genes isknown in the art (Liu et al. (1987) P.N.A.S. 84:3439 and (1987) J.Immunol. 139:3521). mRNA is isolated from a hybridoma or other cellproducing the antibody and used to produce cDNA. The cDNA of interestmay be amplified by the polymerase chain reaction using specific primers(U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library ismade and screened to isolate the sequence of interest. The DNA sequenceencoding the variable region of the antibody is then fused to humanconstant region sequences. The sequences of human constant regions genesmay be found in Kabat et al. (1991) Sequences of Proteins ofImmunological Interest, N.I.H. publication no. 91-3242. Human C regiongenes are readily available from known clones. The choice of isotypewill be guided by the desired effector functions, such as complementfixation, or activity in antibody-dependent cellular cytotoxicity.Preferred isotypes are IgG1, IgG3 and IgG4. Either of the human lightchain constant regions, kappa or lambda, may be used. The chimeric,humanized antibody is then expressed by conventional methods.

Antibody fragments, such as Fv, F(ab′)₂ and Fab may be prepared bycleavage of the intact protein, e.g. by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′)₂ fragment would include DNAsequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

Consensus sequences of H and L J regions may be used to designoligonucleotides for use as primers to introduce useful restrictionsites into the J region for subsequent linkage of V region segments tohuman C region segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

Expression vectors include plasmids, retroviruses, YACs, EBV derivedepisomes, and the like. A convenient vector is one that encodes afunctionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The resulting chimeric antibody may be joined toany strong promoter, including retroviral LTRs, e.g. SV-40 earlypromoter, (Okayama et al. (1983) Mol. Cell. Bio. 3:280), Rous sarcomavirus LTR (Gorman et al. (1982) P.N.A.S. 79:6777), and moloney murineleukemia virus LTR (Grosschedl et al. (1985) Cell 41:885); native Igpromoters, etc.

Transgenics

The subject nucleic acids can be used to generate transgenic, non-humanplants or animals or site specific gene modifications in cell lines.Transgenic cells of the subject invention include on or more nucleicacids according to the subject invention present as a transgene, whereincluded within this definition are the parent cells-transformed toinclude the transgene and the progeny thereof. In many embodiments, thetransgenic cells are cells that do not normally harbor or contain anucleic acid according to the subject invention. In those embodimentswhere the transgenic cells do naturally contain the subject nucleicacids, the nucleic acid will be present in the cell in a position otherthan its natural location, i.e. integrated into the genomic material ofthe cell at a non-natural location. Transgenic animals may be madethrough homologous recombination, where the endogenous locus is altered.Alternatively, a nucleic acid construct is randomly integrated into thegenome. Vectors for stable integration include plasmids, retrovirusesand other animal viruses, YACs, and the like.

Transgenic organisms of the subject invention include cells andmulticellular organisms, e.g., plants and animals, that are endogenousknockouts in which expression of the endogenous gene is at least reducedif not eliminated. Transgenic organisms of interest also include cellsand multicellular organisms, e.g., plants and animals, in which theprotein or variants thereof is expressed in cells or tissues where it isnot normally expressed and/or at levels not normally present in suchcells or tissues.

DNA constructs for homologous recombination will comprise at least aportion of the gene of the subject invention, wherein the gene has thedesired genetic modification(s), and includes regions of homology to thetarget locus. DNA constructs for random integration need not includeregions of homology to mediate recombination. Conveniently, markers forpositive and negative selection are included. Methods for generatingcells having targeted gene modifications through homologousrecombination are known in the art. For various techniques fortransfecting mammalian cells, see Keown et al. (1990), Meth. Enzymol.185:527-537.

For embryonic stem (ES) cells, an ES cell line may be employed, orembryonic cells may be obtained freshly from a host, e.g. mouse, rat,guinea pig, etc. Such cells are grown on an appropriatefibroblast-feeder layer or grown in the presence of leukemia inhibitingfactor (LIF). When ES or embryonic cells have been transformed, they maybe used to produce transgenic animals. After transformation, the cellsare plated onto a feeder layer in an appropriate medium. Cellscontaining the construct may be detected by employing a selectivemedium. After sufficient time for colonies to grow, they are picked andanalyzed for the occurrence of homologous recombination or integrationof the construct. Those colonies that are positive may then be used forembryo manipulation and blastocyst injection. Blastocysts are obtainedfrom 4 to 6 week old superovulated females. The ES cells aretrypsinized, and the modified cells are injected into the blastocoel ofthe blastocyst. After injection, the blastocysts are returned to eachuterine horn of pseudopregnant females. Females are then allowed to goto term and the resulting offspring screened for the construct. Byproviding for a different phenotype of the blastocyst and thegenetically modified cells, chimeric progeny can be readily detected.

The chimeric animals are screened for the presence of the modified geneand males and females having the modification are mated to producehomozygous progeny. If the gene alterations cause lethality at somepoint in development, tissues or organs can be maintained as allogeneicor congenic grafts or transplants, or in in vitro culture. Thetransgenic animals may be any non-human mammal, such as laboratoryanimals, domestic animals, etc. The transgenic animals may be used infunctional studies, drug screening, etc. Representative examples of theuse of transgenic animals include those described infra.

Transgenic plants may be produced in a similar manner. Methods ofpreparing transgenic plant cells and plants are described in U.S. Pat.Nos. 5,767,367; 5,750,870; 5,739,409; 5,689,049; 5,689,045; 5,674,731;5,656,466; 5,633,155; 5,629,470; 5,595,896; 5,576,198; 5,538,879;5,484,956; the disclosures of which are herein incorporated byreference. Methods of producing transgenic plants are also reviewed inPlant Biochemistry and Molecular Biology (eds Lea & Leegood, John Wiley& Sons)(1993) pp 275-295. In brief, a suitable plant cell or tissue isharvested, depending on the nature of the plant species. As such, incertain instances, protoplasts will be isolated, where such protoplastsmay be isolated from a variety of different plant tissues, e.g. leaf,hypoctyl, root, etc. For protoplast isolation, the harvested cells areincubated in the presence of cellulases in order to remove the cellwall, where the exact incubation conditions vary depending on the typeof plant and/or tissue from which the cell is derived. The resultantprotoplasts are then separated from the resultant cellular debris bysieving and centrifugation. Instead of using protoplasts, embryogenicexplants comprising somatic cells may be used for preparation of thetransgenic host. Following cell or tissue harvesting, exogenous DNA ofinterest is introduced into the plant cells, where a variety ofdifferent techniques are available for such introduction. With isolatedprotoplasts, the opportunity arise for introduction via DNA-mediatedgene transfer protocols, including: incubation of the protoplasts withnaked DNA, e.g. plasmids, comprising the exogenous coding sequence ofinterest in the presence of polyvalent cations, e.g. PEG or PLO; andelectroporation of the protoplasts in the presence of naked DNAcomprising the exogenous sequence of interest. Protoplasts that havesuccessfully taken up the exogenous DNA are then selected, grown into acallus, and ultimately into a transgenic plant through contact with theappropriate amounts and ratios of stimulatory factors, e.g. auxins andcytokinins. With embryogenic explants, a convenient method ofintroducing the exogenous DNA in the target somatic cells is through theuse of particle acceleration or “gene-gun” protocols. The resultantexplants are then allowed to grow into chimera plants, cross-bred andtransgenic progeny are obtained. Instead of the naked DNA approachesdescribed above, another convenient method of producing transgenicplants is Agrobacterium mediated transformation. With Agrobacteriummediated transformation, co-integrative or binary vectors comprising theexogenous DNA are prepared and then introduced into an appropriateAgrobacterium strain, e.g. A. tumefaciens. The resultant bacteria arethen incubated with prepared protoplasts or tissue explants, e.g. leafdisks, and a callus is produced. The callus is then grown underselective conditions, selected and subjected to growth media to induceroot and shoot growth to ultimately produce a transgenic plant.

Utility

The subject chromoproteins and fluorescent mutants thereof find use in avariety of different applications, where the applications necessarilydiffer depending on whether the protein is a chromoprotein or afluorescent protein. Representative uses for each of these types ofproteins will be described below, where the follow described uses aremerely representative and are in no way meant to limit the use of thesubject proteins to those described below.

Chromoproteins

The subject chromoproteins of the present invention find use in avariety of different applications. One application of interest is theuse of the subject proteins as coloring agents which are capable ofimparting color or pigment to a particular composition of matter. Ofparticular interest in certain embodiments are non-toxic chromoproteins.The subject chromoproteins may be incorporated into a variety ofdifferent compositions of matter, where representative compositions ofmatter include: food compositions, pharmaceuticals, cosmetics, livingorganisms, e.g., animals and plants, and the like. Where used as acoloring agent or pigment, a sufficient amount of the chromoprotein isincorporated into the composition of matter to impart the desired coloror pigment thereto. The chromoprotein may be incorporated into thecomposition of matter using any convenient protocol, where theparticular protocol employed will necessarily depend, at least in part,on the nature of the composition of matter to be colored. Protocols thatmay be employed include, but are not limited to: blending, diffusion,friction, spraying, injection, tattooing, and the like.

The chromoproteins may also find use as labels in analyte detectionassays, e.g., assays for biological analytes of interest. For example,the chromoproteins may be incorporated into adducts with analytespecific antibodies or binding fragments thereof and subsequentlyemployed in immunoassays for analytes of interest in a complex sample,as described in U.S. Pat. No. 4,302,536; the disclosure of which isherein incorporated by reference. Instead of antibodies or bindingfragments thereof, the subject chromoproteins or chromogenic fragmentsthereof may be conjugated to ligands that specifically bind to ananalyte of interest, or other moieties, growth factors, hormones, andthe like; as is readily apparent to those of skill in the art.

In yet other embodiments, the subject chromoproteins-may-be used asselectable markers in recombinant DNA applications, e.g., the productionof transgenic cells and organisms, as described above. As such, one canengineer a particular transgenic production protocol to employexpression of the subject chromoproteins as a selectable marker, eitherfor a successful or unsuccessful protocol. Thus, appearance of the colorof the subject chromoprotein in the phenotype of the transgenic organismproduced by a particular process can be used to indicate that theparticular organism successfully harbors the transgene of interest,often integrated in a manner that provides for expression of thetransgene in the organism. When used a selectable marker, a nucleic acidencoding for the subject chromoprotein can be employed in the transgenicgeneration process, where this process is described in greater detailsupra. Particular transgenic organisms of interest where the subjectproteins may be employed as selectable markers include transgenicplants, animals, bacteria, fungi, and the like.

In yet other embodiments, the chromoproteins (and fluorescent proteins)of the subject invention find use in sunscreens, as selective filters,etc., in a manner similar to the uses of the proteins described in WO00/46233.

Fluorescent Proteins

The subject fluorescent proteins of the present invention (as well asother components of the subject invention described above) find use in avariety of different applications, where such applications include, butare not limited to, the following. The first application of interest isthe use of the subject proteins in fluorescence resonance energytransfer (FRET) applications. In these applications, the subjectproteins serve as donor and/or acceptors in combination with a secondfluorescent protein or dye, e.g., a fluorescent protein as described inMatz et al., Nature Biotechnology (October 1999) 17:969-973, a greenfluorescent protein from Aequoria Victoria or fluorescent mutantthereof, e.g., as described in U.S. Pat. Nos. 6,066,476; 6,020,192;5,985,577; 5,976,796; 5,968,750; 5,968,738; 5,958,713; 5,919,445;5,874,304, the disclosures of which are herein incorporated byreference, other fluorescent dyes, e.g., coumarin and its derivatives,e.g. 7-amino-4-methylcoumarin, aminocoumarin, bodipy dyes, such asBodipy FL, cascade blue, fluorescein and its derivatives, e.g.fluorescein isothiocyanate, Oregon green, rhodamine dyes, e.g. texasred, tetramethylrhodamine, eosins and erythrosins, cyanine dyes, e.g.Cy3 and Cy5, macrocyclic chelates of lanthanide ions, e.g. quantum dye,etc., chemilumescent dyes, e.g., luciferases, including those describedin U.S. Pat. Nos. 5,843,746; 5,700,673; 5,674,713; 5,618,722; 5,418,155;5,330,906; 5,229,285; 5,221,623; 5,182,202; the disclosures of which areherein incorporated by reference. Specific examples of where FRET assaysemploying the subject fluorescent proteins may be used include, but arenot limited to: the detection of protein-protein interactions, e.g.,mammalian two-hybrid system, transcription factor dimerization, membraneprotein multimerization, multiprotein complex formation, etc., as abiosensor for a number of different events, where a peptide or proteincovalently links a FRET fluorescent combination including the subjectfluorescent proteins and the linking peptide or protein is, e.g., aprotease specific substrate, e.g., for caspase mediated cleavage, alinker that undergoes conformational change upon receiving a signalwhich increases or decreases FRET, e.g., PKA regulatory domain(cAMP-sensor), phosphorylation, e.g., where there is a phosphorylationsite in the linker or the linker has binding specificity tophosphorylated/dephosphorylated domain of another protein, or the linkerhas Ca²⁺ binding domain. Representative fluorescence resonance energytransfer or FRET applications in which the subject proteins find useinclude, but are not limited to, those described in: U.S. Pat. Nos.6,008,373; 5,998,146; 5,981,200; 5,945,526; 5,945,283; 5,911,952;5,869,255; 5,866,336; 5,863,727; 5,728,528; 5,707,804; 5,688,648;5,439,797; the disclosures of which are herein incorporated byreference.

The subject fluorescent proteins also find use as biosensors inprokaryotic and eukaryotic cells, e.g. as Ca²⁺ ion indicator; as pHindicator, as phorphorylation indicator, as an indicator of other ions,e.g., magnesium, sodium, potassium, chloride and halides. For example,for detection of Ca ion, proteins containing an EF-hand motif are knownto translocate from the cytosol to membranes upon Ca²+ binding. Theseproteins contain a myristoyl group that is buried within the molecule byhydrophobic interactions with other regions of the protein. Binding ofCa²⁺ induces a conformational change exposing the myristoyl group whichthen is available for the insertion into the lipid bilayer (called a“Ca²⁺-myristoyl switch”). Fusion of such a EF-hand containing protein toFluorescent Proteins (FP) could make it an indicator of intracellularCa²⁺ by monitoring the translocation from the cytosol to the plasmamembrane by confocal microscopy. EF-hand proteins suitable for use inthis system include, but are not limited to: recoverin (1-3),calcineurin B, troponin C, visinin, neurocalcin, calmodulin,parvalbumin, and the like. For pH, a system based on hisactophilins maybe employed. Hisactophilins are myristoylated histidine-rich proteinsknown to exist in Dictyostelium. Their binding to actin and acidiclipids is sharply pH-dependent within the range of cytoplasmic pHvariations. In living cells membrane binding seems to override theinteraction of hisactophilins with actin filaments. At pH≦6.5 theylocate to the plasma membrane and nucleus. In contrast, at pH 7.5 theyevenly distribute throughout the cytoplasmic space. This change ofdistribution is reversible and is attributed to histidine clustersexposed in loops on the surface of the molecule. The reversion ofintracellular distribution in the range of cytoplasmic pH variations isin accord with a pK of 6.5 of histidine residues. The cellulardistribution is independent of myristoylation of the protein. By fusingFPs (Fluoresent Proteins) to hisactophilin the intracellulardistribution of the fusion protein can be followed by laser scanning,confocal microscopy or standard fluorescence microscopy. Quantitativefluorescence analysis can be done by performing line scans through cells(laser scanning confocal microscopy) or other electronic data analysis(e.g., using metamorph software (Universal Imaging Corp) and averagingof data collected in a population of cells. Substantial pH-dependentredistribution of hisactophilin-FP from the cytosol to the plasmamembrane occurs within 1-2 min and reaches a steady state level after5-10 min. The reverse reaction takes place on a similar time scale. Assuch, hisactophilin-fluorescent protein fusion protein that acts in ananalogous fashion can be used to monitor cytosolic pH changes in realtime in live mammalian cells. Such methods have use in high throuhgputapplications, e.g., in the measurement of pH changes as consequence ofgrowth factor receptor activation (e.g. epithelial or platelet-derivedgrowth factor) chemotactic stimulation/cell locomotion, in the detectionof intracellular pH changes as-second messenger, in the monitoring ofintracellular pH in pH manipulating experiments, and the like. Fordetection of PKC activity, the reporter system exploits the fact that amolecule called MARCKS (myristoylated alanine-rich C kinase substrate)is a PKC substrate. It is anchored to the plasma membrane viamyristoylation and a stretch of positively charged amino acids(ED-domain) that bind to the negatively charged plasma membrane viaelectrostatic interactions. Upon PKC activation the ED-domain becomesphosphorylated by PKC, thereby becoming negatively charged, and as aconsequence of electrostatic repulsion MARCKS translocates from theplasma membrane to the cytoplasm (called the “myristoyl-electrostaticswitch”). Fusion of the N-terminus of MARCKS ranging from themyristoylation motif to the ED-domain of MARCKS to fluorescent proteinsof the present invention makes the above a detector system for PKCactivity. When phosphorylated by PKC, the fusion protein translocatesfrom the plasma membrane to the cytosol. This translocation is followedby standard fluorescence microscopy or confocal microscopy e.g. usingthe Cellomics technology or other High Content Screening systems (e.g.Universal Imaging Corp./Becton Dickinson). The above reporter system hasapplication in High Content Screening, e.g., screening for PKCinhibitors, and as an indicator for PKC activity in many screeningscenarios for potential reagents interfering with this signaltransduction pathway. Methods of using fluorescent proteins asbiosensors also include those described in U.S. Pat. Nos. 972,638;5,824,485 and 5,650,135 (as well as the references cited therein) thedisclosures of which are herein incorporated by reference.

The subject fluorescent proteins also find use in applications involvingthe automated screening of arrays of cells expressing fluorescentreporting groups by using microscopic imaging and electronic analysis.Screening can be used for drug discovery and in the field of functionalgenomics: e.g., where the subject proteins are used as markers of wholecells to detect changes in multicellular reorganization and migration,e.g., formation of multicellular tubules (blood vessel formation) byendothelial cells, migration of cells through Fluoroblok Insert System(Becton Dickinson Co.), wound healing, neurite outgrowth, etc.; wherethe proteins are used as markers fused to peptides (e.g., targetingsequences) and proteins that allow-the-detection of change ofintracellular location as indicator for cellular activity, for example:signal transduction, such as kinase and transcription factortranslocation upon stimuli, such as protein kinase C, protein kinase A,transcription factor NFkB, and NFAT; cell cycle proteins, such as cyclinA, cyclin B1 and cyclinE; protease cleavage with subsequent movement ofcleaved substrate, phospholipids, with markers for intracellularstructures such as endoplasmic reticulum, Golgi apparatus, mitochondria,peroxisomes, nucleus, nucleoli, plasma membrane, histones, endosomes,lysosomes, microtubules, actin) as tools for High Content Screening:co-localization of other fluorescent fusion proteins with theselocalization markers as indicators of movements of intracellularfluorescent fusion proteins/peptides or as marker alone; and the like.Examples of applications involving the automated screening of arrays ofcells in which the subject fluorescent proteins find use include: U.S.Pat. No. 5,989,835; as well as WO/0017624; WO 00/26408; WO 00/17643; andWO 00/03246; the disclosures of which are herein incorporated byreference.

The subject fluorescent proteins also find use in high through-putscreening assays. The subject fluorescent proteins are stable proteinswith half-lives of more than 24 h. Also provided are destabilizedversions of the subject fluorescent proteins with shorter half-livesthat can be used as transcription reporters for drug discovery. Forexample, a protein according to the subject invention can be fused witha putative proteolytic signal sequence derived from a protein withshorter half-life, e.g., PEST sequence from the mouse ornithinedecarboxylase gene, mouse cyclin B1 destruction box and ubiquitin, etc.For a description of destabilized proteins and vectors that can beemployed to produce the same, see e.g., U.S. Pat. No. 6,130,313; thedisclosure of which is herein incorporated by reference. Promoters insignal transduction pathways can be detected using destabilized versionsof the subject fluorescent proteins for drug screening, e.g., AP1, NFAT,NFkB, Smad, STAT, p53, E2F, Rb, myc, CRE, ER, GR and TRE, and the like.

The subject proteins can be used as second messenger detectors, e.g., byfusing the subject proteins to specific domains: e.g., PKCgamma Cabinding domain, PKCgamma DAG binding domain, SH2 domain and SH3 domain,etc.

Secreted forms of the subject proteins can be prepared, e.g. by fusingsecreted leading sequences to the subject proteins to construct secretedforms of the subject proteins, which in turn can be used in a variety ofdifferent applications.

The subject proteins also find use in fluorescence activated cellsorting applications. In such applications, the subject fluorescentprotein is used as a label to mark a population of cells and theresulting labeled population of cells is then sorted with a fluorescentactivated cell sorting device, as is known in the art. FACS methods aredescribed in U.S. Pat. Nos. 5,968,738 and 5,804,387; the disclosures ofwhich are herein incorporated by reference.

The subject proteins also find use as in vivo marker in animals (e.g.,transgenic animals). For example, expression of the subject protein canbe driven by tissue specific promoters, where such methods find use inresearch for gene therapy, e.g., testing efficiency of transgenicexpression, among other applications. A representative application offluorescent proteins in transgenic animals that illustrates this classof applications of the subject proteins is found in WO 00/02997, thedisclosure of which is herein incorporated by reference.

Additional applications of the subject proteins include: as markersfollowing injection into cells or animals and in calibration forquantitative measurements (fluorescence and protein); as markers orreporters in oxygen biosensor devices for monitoring cell viability; asmarkers or labels for animals, pets, toys, food, etc.; and the like.

The subject fluorescent proteins also find use in protease cleavageassays. For example, cleavage inactivated fluorescence assays can bedeveloped using the subject proteins, where the subject proteins areengineered to include a protease specific cleavage sequence withoutdestroying the fluorescent character of the protein. Upon cleavage ofthe fluorescent protein by an activated protease fluorescence wouldsharply decrease due to the destruction of a functional chromophor.Alternatively, cleavage activated fluorescence can be developed usingthe subject proteins, where the subject proteins are engineered tocontain an additional spacer sequence in close proximity/or inside thechromophor. This variant would be significantly decreased in itsfluorescent activity, because parts of the functional chromophor wouldbe divided by the spacer. The spacer would be framed by two identicalprotease specific cleavage sites. Upon cleavage via the activatedprotease the spacer would be cut out and the two residual “subunits” ofthe fluorescent protein would be able to reassemble to generate afunctional fluorescent protein. Both of the above types of applicationcould be developed in assays for a variety of different types ofproteases, e.g., caspases, etc.

The subject proteins can also be used is assays to determine thephospholipid composition in biological membranes. For example, fusionproteins of the subject proteins (or any other kind of covalent ornon-covalent modification of the subject proteins) that allows bindingto specific phospholipids to localize/visualize patterns of phospholipiddistribution in biological membranes also allowing colocalization ofmembrane proteins in specific phospholipid rafts can be accomplishedwith the subject proteins. For example, the PH domain of GRP1 has a highaffinity to phosphatidyl-inositol tri-phosphate (PIP3) but not to PIP2.As such, a fusion protein between the PH domain of GRP1 and the subjectproteins can be constructed to specifically label PIP3 rich areas inbiological membranes.

Yet another application of the subject proteins is as a fluorescenttimer, in which the switch of one fluorescent color to another (e.g.green to red) concomitant with the ageing of the fluorescent protein isused to determine the activation/deactivation of gene expression, e.g.,developmental gene expression, cell cycle dependent gene expression,circadian rhythm specific gene expression, and the like

The antibodies of the subject invention, described above, also find usein a number of applications, including the differentiation of thesubject proteins from other fluorescent proteins.

Kits

Also provided by the subject invention are kits for use in practicingone or more of the above described applications, where the subject kitstypically include elements for making the subject proteins, e.g., aconstruct comprising a vector that includes a coding region for thesubject protein. The subject kit components are typically present in asuitable storage medium, e.g., buffered solution, typically in asuitable container. Also present in the subject kits may be antibodiesto the provided protein. In certain embodiments, the kit comprises aplurality of different vectors each encoding the subject protein, wherethe vectors are designed for expression in different environments and/orunder different conditions, e.g., constitutive expression where thevector includes a strong promoter for expression in mammalian cells, apromoterless vector with a multiple cloning site for custom insertion ofa promoter and tailored expression, etc.

In addition to the above components, the subject kits will furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, etc., on which the information has been recorded.Yet another means that may be present is a website address which may beused via the internet to access the information at a removed site. Anyconvenient means may be present in the kits.

The following examples are offered by way of illustration and not by wayof limitation.

Experimental

I. Introduction

In the following experimental section, we present eleven new GFP-likeproteins.

II. Materials and Methods

A. Collection of Samples

Samples (100-500 mg of tissue) of Montastraea cavernosa, Condylactisgigantea, Scolumia cubensis and Ricordea florida were collected atFlorida Keys Marine Sanctuary (Long Key), under National MarineSanctuary authorization FKNMS-2000-009. The samples were collectedduring night dives, candidate specimens were picked on the basis oftheir appearance under ultraviolet flashlight. Other samples(Dendronephthya sp., Heteractis crispa, Discosoma sp.3, Zoanthus sp. 2)were picked from private seawater aquariums.

B. Cloning and Expression of GFP-Like Proteins

Total RNA was isolated from the tissue samples following the protocoldescribed in Chomczynski, P. & Sacchi, N. (1987) Anal Biochem 162,156-9. Total cDNA was amplified using SMART™ cDNA amplification kit(Clontech). These amplified cDNA samples were used to amplify3′-fragments of cDNAs coding for GFP-like proteins and then obtain themissing 5′-flanks, exactly as described in Matz, M. V., Fradkov, A. F.,Labas, Y. A., Savitsky, A. P., Zaraisky, A. G., Markelov, M. L. &Lukyanov, S. A. (1999) Nat Biotechnol 17, 969-73. After determining thecomplete cDNA sequence, the coding regions were amplified using the samecDNA samples as were used to clone the 3′- and 5′-flanks as templates.An upstream (“N-terminal”) primer had a 5′-heel(5′-tTGAtTGAtTGAAGGAGAaatatc) carrying stop codons (bold) in all framesand bacterial ribosome-binding site (underlined), followed by the targetcDNA sequence (20-22 bases) starting with initiation codon of the ORF.The downstream (“C-terminal”) primer was 22-25 bases long andcorresponded to the antisense sequence of cDNA around the stop codon ofthe ORF. The resulting fragments were cloned using PGEM-T vector cloningkit (Promega) following the manufacturer's protocol, using Escherichiacoli JM109 strain as host. The colonies were grown onLB/agar/carbenicillin plates supplemented with 0.3 mM IPTG for 16-20hours at 37° C., and then incubated for two days at 4° C. Thefluorescent colonies were selected using fluorescent microscope andstreaked widely on new plates. The same colonies were used for overnightculture inoculation followed by plasmid isolation and sequencing, toconfirm the identity of the clone. The bacteria were harvested from theplates, suspended in 1 ml of PBS and disrupted by sonication. The lysatewas cleared by centrifugation, and its fluorescent properties weredetermined using LS-50B spectrofluorometer (Perkin Elmer Instruments).For mcavRFP and rfloGFP, the early” samples were harvested after 24hours at 37° C., “late” samples—after 24 hours at 37 C followed by fourdays at 4° C.

C. Phylogenetic analysis

The alignment of GFP-like proteins (see supplemental data) wasconstructed after Matz, M. V., Fradkov, A. F., Labas, Y. A., Savitsky,A. P., Zaraisky, A. G., Markelov, M. L. & Lukyanov, S. A. (1999) NatBiotechnol 17, 969-73 taking in account constraints of the proteinstructure. Then the DNA alignment was made following the proteinalignment; excluding the poorly aligned N- and C-terminal regions. Thephylogenetic tree was constructed using Tree-Puzzle software (Strimmer,K. & von Haeseler, A. (1996) Mol. Biol. Evol. 13, 964-969) under HKYmodel of DNA evolution (Hasegawa, M., Kishino, H. & Yano, K. (1985) J.Mol. Evol. 22, 160-174), assuming that the variability of sites followsgamma-distribution with alpha parameter estimated from the dataset. Thetree was confirmed to be the maximum likelihood tree by PAML software(Yang, Z. (2000) (University College(http://abacus.gene.ucl.ac.uk/software/paml.html), London, England))under REV model (Yang, Z. H., Goldman, N. & Friday, A. (1994) MolecularBiology and Evolution 11, 316-324). The tree built by Tree-Puzzle fromprotein alignment (JTT model, (Jones, D. T., Taylor, W. R. & Thornton,J. M. (1992) CABIOS 8, 275-282) had the same topology but lower supportvalues due to smaller number of informative sites in the proteinalignment.

III. Results and Discussion

A. Nomenclature

For the sake of clarity of phylogenetic analysis representation, in thispaper we are using new nomenclature for GFP-like proteins. Our proteinidentification tags include four-letter leader composed of first letterof genus name and three initial letters of species name, followed bydefinition of color type: GFP—green, RFP—red, YFP—yellow,CP—chromoprotein (non-fluorescent). When the species is not defined, theleader is four initial letters of the genus name. In the case ofmultiple non-identified species of the same genus, a number is added tothe leader (such as in dis3GFP or zoan2RFP); in the case of severalproteins of the same color type found in the same species, the number isadded to the color definition (such as in scubGFP1 and scubGFP2). ForAequorea victoria GFP and drFP583 from Discosoma sp., widely acceptedcommon names are kept: GFP and DsRed.

B. New GFP-like proteins

A total of fourteen new GFP-like proteins were cloned andspectroscopically characterized. The spectral features of 11 of theseproteins are summarized in Table 1 appearing in the figures, as well asthe other figures of the application

This subset of 11 includes representatives exhibiting features not seenbefore in Anthozoan GFP-like proteins. Two green proteins fromCondylactis gigantea (cgigGFP) and Heteractis crispa (hcriGFP) possessdouble-peaked excitation spectra very similar to the one of wild-typeGFP, suggesting that their chromophores undergo photoconversion betweenneutral and ionized states (Brejc, K., Sixma, T. K., Kifts, P. A., Kain,S. R., Tsien, R. Y., Ormo, M. & Remington, S. J. (1997) Proc Natl AcadSci USA 94, 2306-11; Palm, G. J., Zdanov, A., Gaitanaris, G. A.,Stauber, R., Pavlakis, G. N. & Wlodawer, A. (1997) Nat Struct Biol 4,361-5). The red-emitting protein zoan2RFP, although being very similarto DsRed in the shape of excitation/emission curves, behaves like“timer”: it turns green at first and then matures into red (FIG. 1, Aand B), similarly to one of the mutant variants of DsRed (Terskikh, A.,Fradkov, A., Ermakova, G., Zaraisky, A., Tan, P., Kajava, A. V., Zhao,X., Lukyanov, S., Matz, M., Kim, S., Weissman, I. & Siebert, P. (2000)Science 290, 1585-8.). The two new red-emitters from great star coralMontastraea cavernosa (mcavRFP) and florida corallimorph Ricordeaflorida (rfloRFP) also show a “timer” phenotype (FIG. 1, C-F). Incontrast to zoan2RFP, they failed to mature completely into red in ourbacterial expression trials, which resulted in two-peak emission spectrasuch as shown in FIG. 1 (D and F). Remarkably, for both these proteins,the red emission band in the more mature form had major excitation peakvirtually identical to the one of the immature green form, theyellow-orange excitation peak being significantly smaller (FIG. 2). Thisis strikingly different from the rest of the orange-red proteins, inwhich the red emission is excited best in yellow-orange region (FIG. 4,Table 1, spectra E). This unusual shape of excitation spectra may be dueto photoconversion of the ionization states of the chromophore (byanalogy with green proteins), or to even more profound differences inthe chromophore structure. In favor of the latter speaks the fact thatthe shape of the red emission peaks of mcavRFP and rfloRFP is notablydifferent from other orange-red proteins: it is much narrower and almostsymmetrical in contrast to the wide and skewed emission peak of theothers (compare spectra E and F in Table 1, FIG. 4). Meanwhile, in GFPfrom Aequorea victoria, presence or absence of photoconversion does nothave much effect on the shape of emission spectra (Heim, R., Cubitt, A.B. & Tsien, R. Y. (1995) Nature 373, 663-4). The striking similarity ofmajor excitation peaks for mature and immature proteins makes ittempting to suggest that in mcavRFP and rfloRFP, the “built-in”fluorescence resonance energy transfer (FRET) from immature green formof the protein to the mature red form is the major mechanism giving riseto red emission.

C. Structural/Spectral Types of GFP-Like Proteins

In our view, the best way to classify GFP-like proteins is by theircolor as it appears to human eye. We discriminate four color types ofGFP-like proteins: green, yellow, orange-red and purple-blue, orchromoproteins (Table 1, FIG. 14). All of them share the same fold ofpolypeptide chain, termed “beta-can” (Ormo, M., Cubift, A. B., Kallio,K., Gross, L. A., Tsien, R. Y. & Remington, S. J. (1996) Science 273,1392-5.; Yang, F., Moss, L. G. & Phillips, G. N., Jr. (1996) NatBiotechnol 14, 1246-51). However, there are substantial differencesbetween these color types as far as the chromophore structure isconcerned (see Table 1). In GFP (green color), the chromophore is formedby residues 65-67 (Ser-Tyr-Gly) as a result of condensation between thecarbonyl carbon of Ser-65 and the amino nitrogen of Gly-67 that producesa five-member ring, followed by the dehydrogenation of the Tyr-66methylene bridge. All the green proteins apparently possess the samechromophore, and the differences in the spectral shapes are explained bymodifications of its environment. It must be noted that the greenproteins having excitation/emission spectra such as on panel A on Table1 are sometimes called cyan or even blue, but to the human eye the colorof these proteins after purification still appears bright green. In thered protein DsRed, the chromophore synthesis includes one more stagethat extends the conjugated pi-system of the chromophore—dehydrogenationof the bond between the alpha carbon and amino nitrogen of the firstchromophore-forming residue. Meanwhile, in the chromoproteinsrepresentative asulCP, cyclization leads to the formation of asix-member rather than five-member ring, and the critical step increating the extended conjugated pi-system is breakage of thepolypeptide chain immediately before the chromophore. Notably, no otherchromoprotein contains such a chain break, as demonstrated by denaturingelectrophoresis of the bacterial exprssion products (data not shown).This indicates that the chromophore structure of asulCP is exceptionrather than the rule within this color type. Biochemical and mutagenesisstudies of the yellow zoanYFP indicated that this protein has yetanother chromophore structure. So, it must be concluded that althoughpronounced color difference between GFP-like proteins indicatesdifference in chromophore structures (which makes it reasonable to usecolor for classification), different chromophores might be found even inthe proteins of the same color, as it happens within the group ofchromoproteins and probably within the orange-red group.

D. Molecular basis of color conversion

Since a chromophore synthesis pathway in DsRed is an extended form ofthe GFP pathway, it can be easily imagined that any mutation damagingthe additional autocatalytic stage in DsRed would convert it into greenprotein. Indeed, at least seven different mutant variants of DsRedemitting in the green range were found during random and site specificmutagenesis. Similar reasoning should apply to the two new red proteins,because their red emission also arises as a result of furthermodification of the green-emitting chromophore.

It has been shown that a single amino acid replacement can convert achromoprotein into a DsRed-like red fluorescent protein. It isparticularly unexpected for asulCP from Anemonia sulcata, which has beendirectly demonstrated to contain a very dissimilar chromophore; and itstill seems unlikely that its red fluorescent mutant variant actuallyswitches to synthesizing a DsRed-type chromophore instead of originalone. However, random mutations in this mutant variant resulted inappearance of green-emitting forms. Since no green-emitting intermediatestage was present in the original asulCP autocatalytic pathway,formation of green-emitting structure in these mutants signifies asubstantial deviation, most probably towards a GFP/DsRed type ofchromophore formation sequence judging by the shape ofexcitation/emission spectra of the green asulCP mutants.

Finally, yellow protein zoanYFP also can be converted intogreen-emitting state by at least two different amino acid replacements.

Taking these data into account, the following explanation of theobserved phylogenetic pattern seems plausible: that differentchromophore structures, even the most dissimilar ones, are alternativeproducts synthesized with the help of a basically similar autocatalyticenvironment, rather than outcomes of prolonged evolution of differentcatalytic mechanisms. Apparently, just a few amino acid changes in theprotein may act like a switch between alternative pathways, asexemplified by mutagenesis results on asulCP chromoprotein.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1. A nucleic acid having a sequence of residues that is substantiallythe same as or identical to a nucleotide sequence of at least 10residues in length of SEQ ID NOS:01, 03, 05, 07, 09, 11, 13, 15, 17, 19,21, 23, 25 or
 27. 2. The nucleic acid according to claim 1, wherein saidnucleic acid has a sequence similarity of at least about 60% with asequence of at least 10 residues in length of SEQ ID NOS: 01, 03, 05,07, 09, 11, 13, 15, 17, 19, 21, 23, 25 or
 27. 3. A nucleic acid presentin other than its natural environment that encodes a chromo and/orfluorescent protein that has an amino acid sequence of: SEQ ID NOS: 02,04, 06, 08, 10, 12, 14, 16, 18, 20, 22, 24, 26 or
 28. 4. A nucleic acidthat encodes a mutant protein of a protein that has an amino acidsequence of: SEQ ID NOS: 02, 04, 06, 08, 10, 12, 14, 16, 18, 20, 22, 24,26 or
 28. 5. The nucleic acid according to claim 4, wherein said mutantprotein comprises at least one point mutation as compared to its wildtype protein.
 6. The nucleic acid according to claim 4, wherein saidmutant protein comprises at least one deletion mutation as compared toits wild type protein.
 7. A fragment of the nucleic acid according toclaim
 1. 8. An isolated nucleic acid or mimetic thereof that hybridizesunder stringent conditions to a nucleic acid according to claim
 1. 9. Aconstruct comprising a vector and a nucleic acid according to claim 1.10. An expression cassette comprising: (a) a transcriptional initiationregion functional in an expression host; (b) a nucleic acid according toclaim 1; and (c) and a transcriptional termination region functional insaid expression host.
 11. A cell, or the progeny thereof, comprising anexpression cassette according to claim 10 as part of an extrachromosomalelement or integrated into the genome of a host cell as a result ofintroduction of said expression cassette into said host cell.
 12. Amethod of producing a chromo and/or fluorescent protein, said methodcomprising: growing a cell according to claim 11, whereby said proteinis expressed; and isolating said protein substantially free of otherproteins.
 13. A protein or fragment thereof encoded by a nucleic acidaccording to claim
 1. 14. An antibody binding specifically to a proteinaccording to claim
 13. 15. A transgenic cell or the progeny thereofcomprising a transgene comprising a nucleic acid according to claim 1.16. A transgenic organism comprising a transgene comprising a nucleicacid according to claim
 1. 17. In an application that employs a chromo-or fluorescent protein, the improvement comprising: employing a proteinaccording to claim
 13. 18. In an application that employs a nucleic acidencoding a chromo- or fluorescent protein, the improvement comprising:employing a nucleic acid according to claim
 1. 19. A kit comprising anucleic acid according to claim 1 and instructions for using saidnucleic acid.